Synopsis The population-based association between low vitamin D status and increased cancer risk can be inconsistent but is now generally accepted. These relationships link low serum 25 hydroxyvitamin D levels to cancer while cell-based studies show that the metabolite 1,25 dihydroxyvitamin D is the biologically active metabolite that works through vitamin D receptor to regulate gene transcription. Here we review the literature relevant to the molecular events that may account for the beneficial impact of vitamin D on cancer prevention or treatment. This data shows that while vitamin D-induced growth arrest and apoptosis of tumor cells or their non-neoplastic progenitors are plausible mechanisms, other chemoprotective mechanisms are also worthy of consideration. These alternative mechanisms include enhancing DNA repair, antioxidant protection, and immunomodulation. In addition, other cell targets such as the stromal cells, endothelial cells, and cells of the immune system may be regulated by 1,25 dihydroxyvitamin D and contribute to vitamin D mediated cancer prevention.

Genetically modified mice have been used for colon cancer research, but findings from these models are confounded by expression of cancer in multiple organs. We sought to create a transgenic mouse with Cre recombinase (Cre) expression limited to the epithelial cells of the large intestine and used this model to study colon cancer driven by adenomatosis polyposis coli (APC) gene inactivation. A promoter/enhancer from the mouse carbonic anhydrase

We studied the effect of prolonged activation of Mitogen Activated Protein Kinase (MAPK) signaling on 1,25 dihydroxyvitamin D (1,25(OH)2D3)-action in the immortalized human prostate epithelial cell line RWPE1 and its K-Ras transformed clone RWPE2. 1,25(OH)2D3-treatment caused growth arrest and induced gene expression in both cell lines but the response was blunted in RWPE2 cells. Vitamin D receptor (VDR) levels were lower in RWPE2 cells but VDR over-expression did not increase vitamin D-mediated gene transcription in either cell line. In contrast, MAPK inhibition restored normal vitamin D transcriptional responses in RWPE2 cells and MAPK activation with constitutively active MEK1R4F reduced vitamin D-regulated transcription in RWPE1 cells. 1,25(OH)2D3-mediated transcription depends upon the VDR and its heterodimeric partner the Retinoid X Receptor (RXR) so we studied whether changes in the VDR-RXR transcription complex occur in response to MAPK activation. Mutation of putative phosphorylation sites in the Activation Function 1 (AF-1) domain (S32A, T82A) of RXRα restored 1,25(OH)2D3-mediated transactivation in RWPE2 cells. Mammalian two-hybrid and co-immunoprecipitation assays revealed a vitamin D-independent interaction between Steroid Receptor Coactivator-1 (SRC-1) and RXRα that was reduced by MAPK activation and was restored in RWPE2 cells by mutating S32 and T82 in the RXRα AF-1 domain. Our data show that a common contributor to cancer development, prolonged activation of MAPK signaling, impairs 1,25(OH)2D3-mediated transcription in prostate epithelial cells. This is due in part to the phosphorylation of critical amino acids in the RXRα AF-1 domain and impaired co-activator recruitment.

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-toglutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection.IMPORTANCE Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

The papillomavirus (PV) E2 protein coordinates viral transcription and genome replication. Following a strategy to identify amino acids in E2 that are posttranslationally modified, we reported that tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) complexes with and phosphorylates E2, which inhibits viral DNA replication. Here, we present several lines of evidence indicating that tyrosine (Y) 138 of HPV-31 E2 is a substrate of FGFR3. The active form of FGFR3 bound to and phosphorylated the region of amino acids (aa) 107 to 175 in HPV-31 E2. The E2 phenylalanine (F) mutant Y138F displayed reduced FGFR3-induced phosphotyrosine. A constitutive kinase-active FGFR3 inhibited wild-type (WT) E2-induced E1-dependent DNA replication, while the 138F mutant retained activity. The tyrosine to glutamic acid (E) mutant Y138E, which can mimic phosphotyrosine, failed to induce transient DNA replication, although it maintained the ability to bind and localize the viral DNA helicase E1 to the viral origin. The bromodomain-containing protein 4 (Brd4) binds to E2 and is necessary for initiation of viral DNA synthesis. Interestingly, the Y138E protein coimmunoprecipitated with full-length Brd4 but was defective for association with its C-terminal domain (CTD). These results imply that the activity of the FGFR3 kinase in the infected epithelial cell restricts the HPV replication program through phosphorylation of E2 at Y138, which interferes with E2 binding to the Brd4 CTD, and that this interaction is required for initiation of viral DNA synthesis. IMPORTANCE Human papillomaviruses (HPVs) are highly infectious pathogens that commonly infect the oropharynx and uterine cervix. The idea that posttranslational modifications of viral proteins coordinates viral genome replication is less explored. We recently discovered that fibroblast growth factor receptor 3 (FGFR3) phosphorylates the viral E2 protein. The current study demonstrates that FGFR3 phosphorylates E2 at tyrosine 138, which inhibits association with the C-terminal peptide of Brd4. This study illustrates a novel regulatory mechanism of virus-host interaction and provides insight into the role of Brd4 in viral replication.